Substrate effect on α-chymotrypsin activity in aqueous solutions of “big-head” ammonium salts

The effect of two ammonium salts with a bulky head group, the tetrabutylammonium bromide (TBABr) and the surfactant cetyltributylammonium bromide (CTBABr) on α-chymotrypsin hydrolysis rate toward three peptidyl substrates was investigated. N-Succinyl-l-phenylalanine-p-nitroanilide (SPpNA), N-succinyl-l-alanyl-l-alanyl-l-phenylalanine-p-nitroanilide (SAAPpNA) and N-succinyl-l-alanyl-l-alanyl-l-prolyl-l-phenylalanine-p-nitroanilide (SAAPPpNA), which contain the same chromogenic and N-protecting groups, but a different number of amino acidic residues, were selected.The relative activity showed a bell-shaped dependence on additive concentration, with a maximum which ranged from 1 × 10−3 M to 5 × 10−3 M for CTBABr and at 0.3 M for TBABr. Both the additives induced α-chymotrypsin superactivity, but their effect decreased as the peptide chain length of the substrate increased.Analysis of the kinetic parameters indicated that the activation was mainly due to an increase in kcat values, probably caused by enzyme conformational changes induced by the additives, while KM remained almost unchanged. The very notable effect of both CTBABr and TBABr on SPpNA hydrolysis rate and the limited activation with SAAPpNA and SAAPPpNA could be probably due to non-specific interactions between the additives and the subsites next to the catalytic point within α-chymotrypsin active site, not allowed with the longer amino acidic chain substrates.